Ehrlichiae are obligate intracellular gram-negative bacteria that replicate in leukocytes. Several Ehrlichia spp. are now known to cause emerging vector-borne zoonoses in the United States, including a new granulocytotropic ehrlichia, the human granulocytic ehrlichiosis (HGE) agent, and Ehrlichia chaffeensis, a new monocytotropic ehrlichia causes human monocytic ehrlichiosis. E. sennetsu is another monocytotropic agent and the first human ehrlichial pathogen discovered. These organisms often cause severe systemic febrile illness accompanied by hematological abnormalities. The mechanisms responsible for pathogenesis, and the development of protective immunity are poorly understood. The research progress on this group of bacteria has been hampered because cultivation and purification of ehrlichiae are difficult. As these organisms are obligate intracellular pathogens, conventional microbiological and molecular genetic approaches have not been feasible. The overall goal of the proposed study is to sequence and annotate the genome of three human pathogens: the HGE agent, E. chaffeensis and E. sennetsu, each representing three genetically divergent groups of all Ehrlichia spp. (up to 15 percent divergent in 16S rRNA gene sequences). The genomes of the HGE agent, E. chaffeensis and E. sennetsu are 1.5, 1.2 and 0.9 Mb in size, respectively. Thus, genome sequencing of these organisms will yield immediate answers to a number of important questions. For example, it will be possible to identify which metabolic activities are present, whether lipid A or LPS is present, and what types of iron uptake system exists in Ehrlichia spp. The rationale for choosing three representative Ehrlichia species is that they are sufficiently different both with respect to genome issues as well as the interaction with the host cells to warrant a complete comparative analysis of all three genomes. A whole genome shotgun approach will be employed. Additionally, a large fragment library will be prepared in a BAC vector to serve as scaffolding for the ordering of contigs and for gap closure. Annotation of the assembled sequence will be performed after assembly. Genomes will be compared to determine the metabolic capabilities of each organism as well as to characterize unique and conserved virulence determinants. Sequencing of these three Ehrlichia spp. will provide comprehensive and comparative knowledge on ehrlichial organisms. The data to be obtained along with data accumulating from projects on other obligate intracellular bacteria and vector-borne pathogens will facilitate the development of new testable hypotheses regarding the virulence mechanisms and the development of a rational vaccine candidate, chemotherapy, and differential diagnostic methods.